HUMAN PARVOVIRUS PEPTIDES WITH DISULFIDE BRIDGE FOR IMMUNIZATION OR DIAGNOSIS

PEPTIDES DU PARVOVIRUS HUMAIN A PONT DISULFURE A DES FINS D'IMMUNISATION OU DE DIAGNOSTIC

English Abstract

2053240 9013567 PCTABS00002
An artificial peptide having an amino acid sequence which
corresponds to a naturally occurring amino acid sequence of a human
parvovirus comprising an epitope and which further has two cysteine
residues located on each side of said epitope, and further having
a sulphur bridge between said two cysteine residues, which has
been formed by a chemical oxidation step, is described.
Furthermore, an artificial antigen which reacts with antibodies induced by a
human parvovirus is described. Said antigen mainly consists of
an artificial peptide according to the invention. Additionally, a
method of detecting antibodies induced by a human parvovirus in a
sample of body fluid, wherein said sample is subjected to an
immunoassay, especially ELISA, and wherein an artificial antigen
according to the invention is used as a diagnostic antigen, is
described. A diagnostic immunoassay kit for said method is also
described. Finally, a vaccine composition comprising, as an immunizing
component, at least one antigen according to the invention, is
described.

Claims

WO 90/13567 PCT/SE90/00276
11
CLAIMS
1. An artificial peptide having an amino acid se-
quence which corresponds to a naturally occurring amino
acid sequence of a human parvovirus comprising an epitope
and which further has two cysteine residues located
on each side of said epitope, c h a r a c t e r i s e d
in that the peptide has a sulphur bridge between said
two cysteine residues which has been formed by a chemical
oxidation step.
2. An artificial peptide according to claim 1,
c h a r a c t e r i s e d in that it is chosen from
the group consisting of
the peptide having the modified amino acid sequence
<IMG>
and peptides having a shorter sequence thereof including
the modified sequence
<IMG> ;
and the peptide having the modified amino acid sequence
<IMG>
and peptides having a shorter sequence thereof including
the modified sequence

WO 90/13567 PCT/SE90/00276
12
<IMG>;
3. An artificial antigen which reacts with anti-
bodies induced by a human parvovirus, c h a r a c t e -
r i s e d in that the antigen mainly consists of an
artificial peptide having an amino acid sequence which
corresponds to a naturally occurring amino acid sequence
of a human parvovirus comprising an epitope and which
further has two cysteine residues located on each side
of said epitope, and further having a sulphur bridge
between said two cysteine residues which has been formed
by a chemical oxidation step.
4. An artificial antigen according to claim 3,
c h a r a c t e r i g e d in that the antigen mainly
consists of an artificial peptide chosen from the group
consisting of
the peptide having the modified amino acid sequence
<IMG>
and peptides having a shorter sequence thereof including
the modified sequence
<IMG>;
and the peptide having the modified amino acid sequence
<IMG>.

WO 90/13567 PCT/SE90/00276
13
and peptides having a shorter sequence thereof including
the modified sequence
<IMG>
5. An artificial antigen according to claim 3 or
4, c h a r a c t e r i s e d in that it has been immo-
bilized or coupled to a carrier.
6. A method of detecting antibodies induced by
a human parvovirus in a sample of body fluid, wherein
said sample is subjected to an immunoassay, c h a r a c -
t e r i s e d in that an artificial antigen according
to any one of claims 3-5 is used as a diagnostic antigen.
7. A method according to claim 6, wherein said
sample is subjected to enzyme-linked immunosorbent assay
(ELISA) c h a r a c t e r i s e d in that an artifi-
cial antigen according to any one of claims 3-5 is used
as a diagnostic coating antigen.
8. A diagnostic immunoassay kit for the detection
of antibodies induced by a human parvovirus in a sample
of body fluid, c h a r a c t e r i s e d in that it
comprises as a diagnostic antigen an artificial antigen
according to any one of claims 3-5.
9. A diagnostic immunoassay kit according to claim
8, c h a r a c t e r i s e d in that it additionally
comprises
a positive standard serum sample,
a negative standard serum sample,
an enzyme conjugate,
optionally a substrate for the enzyme conjugate,
and optionally buffer solution(s) and/or washing
solution(s).
10. A vaccine composition, c h a r a c t e r i -
s e d in that is comprises as an immunizing component,
at least one antigen selected from the artificial anti-

WO 90/13567 PCT/SE90/00276
14
gens according to any one of claims 3-5, together with
a nontoxic pharmaceutically acceptable carrier and/or
diluent.

Description

~J~ .t~
W090tl3567 PCT/SE90/00276
r,~
, .. .
NEW HUMAN PARYOYIRUS PEP~IDES WITH DISULFIDE BRIOGE
FOR IMMUNIZATION OR DIAGNOSIS
The present invention relates to artificial peptides
having an amino acid sequence which corresponds ta a
naturally occurring amino acid sequence of a human
parvovirus comprising an epi~ope and which ~urther
has two cysteine residuas loca~ed on each side o~
said epitope, and fur~her having a sulphur bridge
between said two cysteine residues which has been
formed by a chemical oxidation step. The invention
also relates to arti~icial antigen5, which react with
antibodies induced by ~ human parvoviru , a method
of detecting antibodies induced by ~ human parvovir~
in a sample o~ body ~luld, a dia~no~tic immunoas~y
kit ~or said metho~, and ~ vaccin@ compos~lon compri-
~ing, a~ an immunizing compon~nt, at l@~st one an~igen
o the invention.
Backqround
A hum~n parvovirus, Bl9, gives rise to erythema
in~ectiosum in children. It is also associated with
aplastic crisis in patients with chronic hemolytic
(e.g. sickle cell) anemia and with spontaneous abortion
intrauterine death or hydrops fetalis when the woman
aquires the infection during pregnancy. The fetus
infection might be cured if the diagnosis is made
early. (J Virol 58: 921 - 936, 1986i Shade et al).
The ~irus can only be cultivated in human bonemarrow,
which hampers isolation for serology. Peptide-based
serology would therefore be a means o~ dia~nosis.
One common way to establish a diagnosis through
antibody detection is to screen serum samples by enæyme-
-link~d immunosorbent aqsay ~ELISA) (Sarn~adharan, M.G.,
Popovic, M., ~ruch, L., Sch~pbach, ~. and Gallo, ~.C.)
Wells o~ microplates coated with viral antigens are
reacted with the serum samples under investigation,
washed, and antihuman I~ add-ed The lat~Qr reag~nt
.. ,.. , .. ".. .. ., . ~ . .... , . . . .............. ., . . ~ . . . .
~ :. .. .. ,. .. , . : , , , .. ;,~ .. . . .. .

W090/l35~7 2 0 ~ 3 2 ~ 3 PcT/sEgn/oo276 ~
is labelled with an enzyme. After washing, the enzyme
labelled antihuman Ig remains only if specific antiviral
Ig was present in the serum sample. It is visualized
by addition of a substrate for the enzyme and the
color reaction quantified in a spectrophotometer.
No assay has so far been developed for detection
of antibodies induced by human parvovirus in a sample
of body fluid.
Description of the invention
The present invention provides i.a. a rapid,
sensitive and specific assay or detection of anti-
bodies induced by a human parvovirus.
In one aspect of the invention there is provided
an artificial peptide having an amino acid sequence
which correspond~ to a na~urally occurring amino acid
sequence oE a human parvoviru~ comprising an ~pitop~
and which ~urkher h~ two cy3t~ina r~8idu~ located
on each sid~ o~ ~aid ~pitopa, and ~urth~r havin~ a
sulphur bridg~ be~weon snid ~wo cy~teln~ r~ldue~
which has bcen eormed by a chemica1 oxldatlon step.
It is believed ~ha~ this stabilization of the peptide
by a sulphur bridge between two cysteine residues
i8 responsible or the useul propertie~ o the peptide,
such as an enhancement of the antibody binding activity,
as well as the chemical stability of the final product.
The artificial peptide includes at least two
cysteine residues, which are cyclized to form a sul-
phur bridge. The two cysteine residues which are linked
together may have one or more amino acid residues
comprising an epitope between themselves, such as
2 to 10 residues. If the artificial peptide according
to the invention includes more than two cysteine resi-
dues, still only one sulphur bridge between two cysteine
residues is ~ormed by a chemical oxidation step.
In a pre~erred embodiment o~ thi~ aspect oE the
inven~ion ther~ is prov.ided an arti~icial p~ptide,
: . : . .. i :. : : : : ~

WO90/13567 PCT/SE90/0027
~hich is chosen from the group consisting of
the peptide having the modified amino acid sequence
lI-Phe-Ser-Pro-Ala-Ala-Ser-Ser-Cys-llis-Asn-Ala-Ser-
I
-Gly-Lys-Glu-A1a-Lys-Val Cys-Thr-Ile-Ser Pro-Ile-N1~2
and peptides having A shorter sequence thereof including
~h~ modiied sequence
-Cys-llis-Asn-Ala-Ser-Gly-Lys-Glu-Ala-Lys-Val-Cys-;
and -the peptide having the modiied amino acid sequence
f~-G~y-Lys-Glu~ Lys-Val-Cy5-~hr~ Scr-Pro-~le-
-Cys-Gly~Tyr-Ser-Thr-Pro-Trp~Arg-Tyr-Leu-N1~2
J
and peptides having a shorter sequence thereoE including
the modified sequence
.':
-Cys-Thr-Ile-Ser-Pro-Ile-Cys-;
In another aspect of the invention there is pro-
vided an artificial antigen which reacts with antibodies
induced by a human parvovirus, which antigen mainly
consists of an artificial peptide having an amino
acid sequence which corresponds to a naturally occurring
; amino acid sequence of a human parvovirus comprising
an epitope and which ~urther has two cy9teine reqidues
located on each side of said epitope, and further
having a sulphur bridge between said two cysteine
. .
,~ ",~, ,, , . : . . . . . . . . . . ` .

WO90/13567 2 ~ 5 ~ 2 ~ a PCT/SE90/00276
residues which has been formed by a chemical oxidation
step
In the specification and claims the expression
"antigen mainly consists of an artificial peptide"
indicates that the ability of the antigen to react
with antibodies derives from the artificial peptide.
In a preferred embodiment of this aspect of the
invention there is provided an arti'ficial antigen
which reacts wi~h an~ibodies induced by a human parvo-
virus, which an~igen m~inly consists o a preferred
arti~icial peptide according to the invention, exem-
plified above.
The artificial antigens according to the invention
can be immobilized or coupled to a carrier, such as
mineral carriers, e.~. aluminium hydroxide, calcium
phosphate, ~tc~, plastic sureaces, e.g. micraplates,
bead~, etc., protein~, such a~ bovine sarum albumin
~r an immunizing component, such a~ k@yhol~ limpat
haemoc~anin.
Even though the arti~icial an~iy~n~ according
to the invention so ar only have been used as diag-
nostic antiqens to detect antibodies induced by a
human parvovirus, in a sample of body ~luid, i~ is
believed that they can be used as immunizing components
in vaccine compositions against a human parvovirus.
Thus, a further aspect of the invention provides
a vaccine composition, which comprises as an immunizing
component, at least one antigen selected from artifi-
cial antigens according to the invention, together
with a nontoxic pharmaceutically acceptable carrier
and/or diluent.
In yet another aspect of the invention there
is provided a method of detecting antibodies induced
by a human parvovirus in a sample of body ~luid, wherein
said sample is subjec~ed to an immunoassay and ~n
arti~icial antigen accordinq to the invention is used
as a diagnostic antigen. Examples of useful body fluids
are urine, saliva, tear fluid, milk, serum, blood
and cerebrospinal ~luid.
:, . ~..... ..... ., . :, ;, .. .... . .

WO 90tl3561 2 ~ ~ 3 2 ~ ~ PCT/SE90~00276
", j,
The immunoassay in ~hich the artificial antisens
according to the invention can be used as diagnostic
antigens is any immunoassay of choice, such as enzyme- '
linked immunosorbent assay (ELISA), radioimmunoassay 1 '
(RIA), immunodiffusion or immunoelectrophoreses (IE).
In a preferred embodiment of this aspect o the
invention ELISA is used as the immunoassay of choice.
In a further aspect of the invention there is
provided a diagnostic immuno~ssay kit for the detection
of antibodies induced by a hum~n p~rvovirus in a sample
o~ b~dy ~luid, wherein an ~rtieicial antigen according
to the invention is included as a diagnostic antigen.
Depending on the immunoassay to be used, the ki~ will
comprise other items, such as a positive standard
serum sample, a negative standard serum s~imple, in
~he c~se o~ EI.ISA an enzyme conjugate and optionaLly
subqkr~te ~or the cnæ~me conju~at~, and al~o aption~lly
bu~er solution~s) ~nd/or washinc~ ~olutlon~s). Option~11y
all the reagent~ .in th~ klt ~ra con~ain@cl ~n ~ap~r~te
sealed te~t tubes or vi~l~ marked wLth 5p@ei~i~ labels.
Synthesis o~ ~he artif lCi 1 peptides of the invention
The artificial peptides of the invention can
be produced by any known method of producing a linear
peptide sequence, such as cloning, degradation, coupling
of one amino acid residue to the next one in liquid
phase or coupling the amino acids to one another starting
with a solid phase (resin) to which the C-terminal
of the first amino acid is coupled, whereupon the
C-terminal of the next amino acid is coupled to the
N-terminal of the first amino acid, etc., finally
releasing the built-up peptide from the solid phase
~so-called Merrifield synthesis). Once the appropriate
linear peptide sequence is ready, it is subjected
to a chemical oxidation step in order to cyclize two
cysteine residues thereo~, whereby a sulphur brid~e
is ~ormed between ~he cy~eine re~idues.
General descrLDtion of synthesis
In the examples below, all peptides were synthe-
sized on an Applied 8iosystems ~30A Peptide Synthesizer
"., ., ,,. . , ... ,. :- ., . . : . -.............. : ,: .................. .
:: : . : : : . ~ :.; . .. : .: , ., . -

W090/13561 ~0~ 3 2 ~ a 6 PCT/SE90/00276
using a double coupling program with termination step
after the second coupling. The resin used was of 4-methyl-
benzhydrylamine type with theoretical loading of 0.66
meq/g (Peptides International, Louisville, KY, USA).
The final product of the synthesis was dried in vacuo
over night. After drying the peptide-resin was suspended
in methanol (70 ml) and saturated with ammonia. The
mixture was placed in pressurized steel vessel and
left over night with magnetic stirring. The resin was
then sep~ra~ed by filtration, washed several times with
mcthanol And thoroughly dried in vacuo. The peptide
was then cleaved rom the resin by treatment with
liquid hydrogen fluorid in the presence of anisole
and ethyl-methyl-sulfide as scavangers (I~F:anisole:EMS
- lO:05:05). After removal of hydrogen fluoride by
evaporation the residue was suspended in ethyl acetate
~lO0 ml) and il~ered. The solid was washed on ~ilter
with additional e~hyl acetate (3xlO0 ml) and th@ cl~av~d
peptide extr~ctec1 with ~c~tlc acid (lO0 ml ) . Th~ extract
was promptl~ diluted to the volume o~ 2 dm3 wi~l1 20~
ac~ic acid in m~hanol and tr~aked with O.l M ~olu~ion
of iodine in methanol until the aint brown colour
persisted. Then the Dowex lx8 ion exchanger in acetate
~or~ was added (3 g) (Bio-Rad, Rlchmond, CA and the
mixture was filtered. The ~iltrate was evaporated
and the residue freeze-dried from 1% acetic acid in
water. The product was then purified by reversed phase
liquid chromatography on a column filled with Vydac
20-25 ~ (Separation Group, CA) in a suitable system
containing acetonitrile in 0.1% trifluoroacetic acid
water solution. The samples collected from the column
were analyzed by analytical I~PLC - Varian 5500 (Sunny-
vale, CA) equipped with BondapaX Cl8 column (Millipore,
Milford, Mass.). Fractions containing pure substance
(>99,5%) were pooled, the solvent was evaporated and
the product freeze-dried from l~ acetic acid in wa~er.
", ., ~ , " , , ~ ,

~,~J~
~090/13567 PCT/SE90/00276
':, 7
The rinal 1~PLC anal~sis was performed on ready product 7
and the structure of the peptide was confirmed by
amino acid analysis and FAB-MS (Fast atom bombardment
spectrometry).
All amino acids used during the synthesis were
protected wlth tert-butoxycarbonyl group at -amino-
function. The side chain protections used were as
follows: Ser(BZL), Thr(BZL), Tyr~2-BrCbz), Lys~2-ClCbz),
Glu(BZL), Arg(Tos), Cys(Mob).
Amino acid derivatives were delivered by Bachem
AG, Switzerland.
The genome o human parvovirus Bl9 encodes two
structural proteins. The sequences corresponding to
VP2 were first synthesized as linear peptides and
then a sulphur bridge between two cysteine residues
were ~or~ed by a che~ical oxidation step.
EXAM
` ~Thc scquenc~ corra~pond~ to a scquenc~ ~ound
in Bl9's ~parvovirus) VP2 recJion).
1~-Phe-Ser-Pro-Ala-Ala-Ser-Ser~Cy9-1liS-~sn-Ala-S~r-Gly-
-Lys-Glu-A1a-Lys~Val-Cys-Thr-Ile-Ser-Pro~le-N1~2
:
The peptide was prepared according to the general
description of synthesis. The structure was confirmed
by aminoacid analysis and by FAB-MS.
EXAMPLE II
--
~The sequence corresponds to a sequence found
in Bl9's (parvovirus) VP2-region).
Ii-Gly-Lys-Glu-Ala-Lys-Val-Cys-Thr-Ile-Ser-Pro-Ile-Cys-
l. . . ~ :
-Gly~Tyr-Ser-Thr-Pro-Trp-Arg-Tyr-Leu-Nll2

WO90/13567 ~ PCT~SE90/00276
The peptide was prepared according to the general
description of synthesis. The structure was confirmed
by aminoacid analysis and by FAB-MS.
Detection of antibodies induced by human par_ovirus
in blood samples
For the detection of antibodies induced by human
parvovirus Bl9 in blood samples use was made of ELISA
~Engvall, E. and Perlmann, P.: Enzyme-linked immuno-
sorbent assay ~ELISA). III Quantification of specific
antiboides by enzyme labelled anti-immunoglobulin
in antigen-coated tubes. J. Immunol. 109.129-135,1972).
Materials:
. .
Plates: Nunc 96 F, Roskilde, Denmark
Conjugate: Alkaline phospha~ase labelled anti~human
~g, Sigma
BufEers: Carbon~k~ buEE~r: 0.05 M sodium carbona~
bu~Qr, pl} approx. 9.5.
Bu~E@r ~: Tryp~in.~z~tion b~e~@r witllou~t
Ca~ and Mg ~, and wi~h 0.5~
bovine serum albumin (BSA) and
0.05~ Tween 20
Diethanolamine, Sigma.
Washing solution: 0.9~ NaCl with 0.05~ Tween 20.
Methods:
-
Coating: A solution of the coating antigen (testpeptide), 5 ~g to 20 ~g per ml, is made in carbonate
buffer. 100 ~1 of the solution is added to each well
of stripes or of 96-well microplates. The adsorption
takes place over night, 18 hrs, at room temperature.
The coated plates can be stored with their contents
in 4C until use.
Serum assay: 1. Empty and wash the plate 4 times
with washing solution.
2. Add 100 ~1 o~ s~rum diluted 1:100
in bu~f~r ~.
3. Add 100 ~1 of just buffer A to one
well; this well serves as a blank.

i3~
O90/13567 PCT/SE9OtO0276
.s~
4. Incubate 2 h at 37C. (Dilute the
conjugate during this period, see
6).
5. Wash 4 times with washing solution.
Empty.
6. Add 100 ~1 of a conjugate, e.g. Alkaline ~
phosphatase labelled anti-human IgG, ` ^
Sigma diluted 1:1500 in bufer A. ;~
7. Incubate at 37C or 2 h. ~Pxepare
sub3trate solution during this period,
see 9),
8. Wash 4 times with washing solution.
Empty.
9. Add 100 ~1 Paranitrophenylphosphate
(2 tablets/10 ml diethanolamine).
Use a clean vessel. 5 min ~re needed
~o dissolve the tablet3.
10. Xncubate 30 min at room temp~xatur~,
S~op the reaction with 25 ~1 5 M
NaOII.
11. Read platc at 405 nm.
Using the above described materials and methods the
~ollowing materials were tested as diagnostic antigens
(coating antigens) ~or the detection of antibodies
induced by human parvovirus Bl9 in serum samples from -
infected patients: ;
; ~A. A region of linear peptides corresponding to
a sequence found in the VP2-region of human parvo- ~-
virus Bl9.~They gave absorbance values of 0.2-0.4
with known~seropositive samples-.
~B. An artificial peptide according to the invention
described in Example I o this specification.
This peptide gave absorbance values oE 1.55 ~ O.q5
when the tes~ was per~Qrm~d on serum samples xom
10 seropositive persons and absorbance values of

~ U
W O 90/1356~ PC~r/SE9Q/00276
0.30 + 0.15 when the test was performed on serum samples
from 9 seronegative persons.
Thus, when the antigen in the test was the peptide
according to the invention the absorbance values of
seropositive sera became significant, and the peptide
showed goo~ reactivity with antibodies induced by
human parvovirus Bl9.